Modeling complete removal of risk assessment questions in the USA predicts the risk of HIV exposure in blood recipients could increase despite the use of nucleic acid testing.

BACKGROUND AND OBJECTIVES: The safety of the blood supply in a number of countries is achieved by interventions that include behaviour-based time-limited or indefinite deferrals and screening of donated units for transfusion-transmitted infections. The relatively high sensitivity of nucleic acid testing (NAT) used in blood donor screening has raised the question of whether such time-based deferrals can be eliminated in favour of individual risk assessment. MATERIALS AND METHODS: Data on the annual number of incident human immunodeficiency virus (HIV) infections associated with various behaviours and on the performance characteristics of NAT applied to donor screening were used to model the number of potentially infected units that might escape detection in the worst-case scenario in which individual risk assessment was implemented, but was not effective as a screening tool, and donors did not otherwise self-select for lower risk. RESULTS: In the absence of effective individual risk-based screening or donor self-selection, the model predicts that in the United States, an additional 39 (95% CI 35-43) HIV-infected units would escape detection by nucleic acid testing, potentially capable of exposing approximately 68 (95% CI 61-75) individuals to the risk of HIV infection through the administration of prepared blood components. CONCLUSION: Despite some inherent uncertainty, the worst-case scenario of completely ineffective individual risk assessment, absence of donor self-selection and increased reliance on NAT for blood screening is estimated to be associated with an approximately fourfold increase in the risk of HIV exposure through transfusion in the United States.

Attachment to fibronectin or vitronectin makes human neutrophil migration sensitive to alterations in cytosolic free calcium concentration.

Transient increases in cytosolic free calcium concentration, [Ca2+]i, appear to be required for the migration of human neutrophils on poly-D-lysine-coated glass in the presence of dilute serum (Marks, P. W., and F. R. Maxfield. 1990. J. Cell Biol. 110:43-52). In contrast, no requirement for [Ca2+]i transients exists when neutrophils migrate on albumin-coated glass in the absence of serum. To determine the mechanism that necessitates [Ca2+]i transients on poly-D-lysine in the presence of serum, migration was examined on substrates consisting of purified adhesive glycoproteins. In the absence of external Ca2+, a treatment which causes the cessation of [Ca2+]i transients, migration on fibronectin (fn) and vitronectin (vn) was significantly inhibited. Migration was also inhibited in Ca2(+)-buffered cells on these substrates, indicating that this effect was the result of an alteration of [Ca2+]i. In the absence of external Ca2+, the inhibition of migration on fn or vn was more pronounced when soluble fn or vn was added to cells migrating on these substrates. This effect of soluble adhesive glycoprotein was specific: in the absence of external Ca2+, soluble fn did not affect the migration of cells on vn, and soluble vn did not affect the migration on fn. No additional inhibition of migration was observed in Ca2(+)-buffered cells with the addition of soluble adhesive glycoprotein. These data indicate that [Ca2+]i transients are involved in continued migration of human neutrophils on fn or vn, proteins which are part of the extracellular matrix that neutrophils encounter in vivo.

Transient increases in cytosolic free calcium appear to be required for the migration of adherent human neutrophils.

Human neutrophils exhibit multiple increases in cytosolic free calcium concentration [( Ca2+]i) spontaneously and in response to the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Jaconi, M. E. E., R. W. Rivest, W. Schlegel, C. B. Wollheim, D. Pittet, and P. D. Lew. 1988. J. Biol. Chem. 263:10557-10560). The function of these repetitive increases in [Ca2+]i, as well as the role of Ca2+ in human neutrophil migration, remain unresolved. We have used microspectrofluorometry to measure [Ca2+]i in single fura-2-loaded human neutrophils as they moved on poly-D-lysine-coated glass in the presence of serum. To investigate the role of Ca2+ in human neutrophil migration, we examined cells in the presence and absence of extracellular Ca2+, as well as intracellular Ca2(+)-buffered and Ca2(+)-depleted cells. In the presence of extracellular Ca2+, multiple increases and decreases in [Ca2+]i were frequently observed, and at least one such transient increase in [Ca2+]i occurred in every moving cell during chemokinesis, chemotaxis, and phagocytosis. In addition, neutrophils that extended pseudopodia and assumed a polarized morphology after plating onto a surface were always observed to exhibit [Ca2+]i transients even in the absence of chemoattractant. In contrast, a [Ca2+]i transient was observed in only one of the nonpolarized stationary cells that were examined (n = 15). Although some cells exhibited relatively periodic increases and decreases in [Ca2+]i, resembling the regular oscillations that have been observed in some cell types, many others exhibited increases and decreases in [Ca2+]i that varied in their timing, magnitude, and duration. Buffering of [Ca2+]i or removal of extracellular Ca2+ damped out or blocked transient increases in [Ca2+]i and reduced or inhibited the migration of neutrophils. Under these conditions, polarized cells were often observed to make repeated attempts at migration, but they remained anchored at their rear. These data suggest that transient increases in [Ca2+]i may be required for the migration of human neutrophils on poly-D-lysine-coated glass in the presence of serum by allowing them to release from previous sites of attachment.

Cdc42 is required for PIP(2)-induced actin polymerization and early development but not for cell viability.

BACKGROUND: Cdc42 and other Rho GTPases are conserved from yeast to humans and are thought to regulate multiple cellular functions by inducing coordinated changes in actin reorganization and by activating signaling pathways leading to specific gene expression. Direct evidence implicating upstream signals and components that regulate Cdc42 activity or for required roles of Cdc42 in activation of downstream protein kinase signaling cascades is minimal, however. Also, whereas genetic analyses have shown that Cdc42 is essential for cell viability in yeast, its potential roles in the growth and development of mammalian cells have not been directly assessed. RESULTS: To elucidate potential functions of Cdc42 mammalian cells, we used gene-targeted mutation to inactivate Cdc42 in mouse embryonic stem (ES) cells and in the mouse germline. Surprisingly, Cdc42-deficient ES cells exhibited normal proliferation and phosphorylation of mitogen- and stress-activated protein kinases. Yet Cdc42 deficiency caused very early embryonic lethality in mice and led to aberrant actin cytoskeletal organization in ES cells. Moreover, extracts from Cdc42-deficient cells failed to support phosphatidylinositol 4,5-bisphosphate (PIP(2))-induced actin polymerization. CONCLUSIONS: Our studies clearly demonstrate that Cdc42 mediates PIP(2)-induced actin assembly, and document a critical and unique role for Cdc42 in this process. Moreover, we conclude that, unexpectedly, Cdc42 is not necessary for viability or proliferation of mammalian early embryonic cells. Cdc42 is, however, absolutely required for early mammalian development.

Tsc2(+/-) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background.

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder in which benign hamartomas develop in multiple organs, caused by mutations in either TSC1 or TSC2. We developed a murine model of Tsc2 disease using a gene targeting approach. Tsc2-null embryos die at embryonic days 9.5-12.5 from hepatic hypoplasia. Tsc2 heterozygotes display 100% incidence of multiple bilateral renal cystadenomas, 50% incidence of liver hemangiomas, and 32% incidence of lung adenomas by 15 months of age. Progression to renal carcinoma, fatal bleeding from the liver hemangiomas, and extremity angiosarcomas all occur at a rate of less than 10%. The renal cystadenomas develop from intercalated cells of the cortical collecting duct and uniformly express gelsolin at high levels, enabling detection of early neoplastic lesions. The tumor expression pattern of the mice is influenced by genetic background, with fewer large renal cystadenomas in the outbred Black Swiss background and more angiosarcomas in 129/SvJae chimeric mice. The slow growth of the tumors in the heterozygote mice matches the limited growth potential of the great majority of TSC hamartomas, and the influence of genetic background on phenotype correlates with the marked variability in expression of TSC seen in patients.

Cofilin and gelsolin segment-1: molecular dynamics simulation and biochemical analysis predict a similar actin binding mode.

An understanding of the actin-depolymerizing function attributed to members of the ADF/cofilin/destrin superfamily requires a structural model of these proteins in complex with actin. As a step toward defining actin-cofilin interactions, the complex of yeast cofilin with monomeric actin was predicted, starting with the actin-gelsolin segment-1 binding mode recently suggested for the actin-destrin complex. After refinement by molecular dynamics simulation, the structure of cofilin converged in a new binding mode that required only minimal changes induced in the actin-cofilin interface. The predicted complex exhibits strong interactions between the N termini of actin and cofilin, mediated by a salt bridge of cofilin Arg3 with actin Asp1. The forming of this salt bridge could be prevented by the phosphorylation of cofilin Ser4, which is believed to inhibit cofilin depolymerization activity. Recent mutagenesis studies, crosslinking experiments and peptide binding studies are consistent with the predicted model of the actin-cofilin complex. The structural homology between cofilin and gelsolin segment-1 binding to actin was confirmed experimentally by two types of competitive binding assays.

Local and global changes in cytosolic free calcium in neutrophils during chemotaxis and phagocytosis.

Neutrophils are capable of undergoing rapid directed movement up a concentration gradient of chemoattractant culminating in the phagocytosis of a target. We have developed a system to make rapid photometric measurements and ratio images of cytosolic free calcium [( Ca2+]i) in human neutrophils loaded with the fluorescent Ca2(+)-sensitive indicator Fura-2 during these processes. In our system neutrophils undergo chemotaxis toward and phagocytosis of IgG and IgM-coated sheep erythrocytes attached to a surface. During chemotaxis and phagocytosis, repetitive transients in [Ca2+]i take place. Accompanying the transients during phagocytosis is a localized [Ca2+]i increase in the periphagosomal region. This localized increase is more apparent in cells phagocytosing particles coated with both IgG and IgM than with IgM alone. No consistent localization of increased [Ca2+]i is seen in cells solely undergoing chemotaxis. The imaging techniques described here allow the observation of [Ca2+]i changes over regions of several microns 2 in a cell with a time resolution of approximately 0.5 s. [Ca2+]i gradients extending over regions greater than approximately 4 microns 2 and lasting at least 1 s can be reliably detected.

Relapsing and remitting severe hypoglycemia due to a monoclonal anti-insulin antibody heralding a case of multiple myeloma.

CONTEXT: We report a novel case of insulin autoimmune syndrome (IAS) presenting with hypoglycemia due to production of a monoclonal anti-insulin antibody in a patient subsequently found to have multiple myeloma (MM). OBJECTIVE: The aim of the study was to describe the 5-yr clinical course of a patient with IAS and MM and to characterize the origin and function of the pathogenic antibody. METHODS: We conducted a longitudinal case history with laboratory investigations to characterize the anti-insulin antibody subtype, specificity, affinity, and origin. RESULTS: The patient presented with IAS, which worsened during treatment of hepatitis C. The patient was then discovered to have a monoclonal gammopathy that progressed to MM. Treatment of the MM induced remission of the neoplasia and IAS, which then followed a synchronized course of progression and response to therapy. An anti-insulin IgG(3)-λ that bound specifically but with low affinity to the insulin B chain (amino acids 9-30) and that was distinct from the primary MM IgG(3)-κ clone was recovered from the patient and cloned. The antibody bound insulin and showed mutations of normal affinity maturation. CONCLUSIONS: We describe a case of MM heralded by IAS, where full characterization of the pathogenic antibody revealed that the monoclonal anti-insulin antibody had originated from a self-reactive clone.

WAG-F8(m1Ycb) rats harboring a factor VIII gene mutation provide a new animal model for hemophilia A.

BACKGROUND: We recently described an inherited coagulopathy arising in an inbred colony of WAG/RijYcb rats. The bleeding phenotype, demonstrated by both male and female rats, included periarticular hemorrhage, spontaneous bruising, prolonged bleeding from minor wounds and maternal peripartum deaths. Coagulation testing of affected rats revealed normal prothrombin time but prolongation of activated partial thromboplastin time to twice that of controls. OBJECTIVE: To determine the specific coagulation factor and the underlying genetic defect responsible for the inherited coagulopathy in the WAG/RijYcb rats. RESULTS: Evaluation of individual clotting factor activities revealed that the affected animals had a specific deficiency of factor (F) VIII (FVIII). The FVIII gene (F8) has an autosomal location on chromosome 18 in rats, in contrast to its location on the X chromosome in mice and humans. Sequencing of F8 cDNA led to the identification of a point mutation resulting in a substitution, Leu176Pro, in the A1 domain, that is predicted to disrupt the tertiary structure of the FVIII molecule. Administration of human plasma or human recombinant FVIII corrects the coagulation abnormality in the affected animals. CONCLUSIONS: We have now identified the genetic basis of the hemostatic defect in the WAG/RijYcb rat colony. The larger size of rats relative to mice and the presence of this coagulation defect in both sexes provide a unique model, well-suited to the development of novel therapies for acquired and hereditary FVIII deficiencies.

Preparation of solutions with free calcium concentration in the nanomolar range using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

There are many uses for solutions with a known free calcium concentration ([Ca2+]free) in the nanomolar range. Most frequently ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) has been used as a buffer for the control of [Ca2+]free; however, under a variety of conditions the use of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) for this purpose would be advantageous. The theory and calculations necessary to make solutions with known [Ca2+]free appropriate for given conditions of pH, ionic strength, and temperature for use with EGTA or BAPTA are reviewed. Practical considerations and methods for making such solutions are detailed. The advantages and disadvantages associated with the use of each of the two chelators are discussed. As one example of the application of solutions with free calcium in the nanomolar range, the dissociation constant of the fluorescent indicator fura-2 for calcium has been determined in a physiologic buffer at 22 and 37 degrees C. For practical reasons, the use of BAPTA is advantageous when solutions with different known [Ca2+]free must be used on a daily basis.

Genomic organization and chromosomal location of murine Cdc42.

cdc42 is a member of the rho family of small GTPases, which are implicated as regulators of cellular morphology. To date, one murine and two human cdc42 isoforms have been identified. Here we report the cloning of a second murine isoform and provide evidence that the two isoforms arise from a single gene by alternative splicing. In contrast with the previously identified murine cdc42 sequence, which is expressed in a wide variety of tissues, the second isoform appears to be expressed exclusively in brain. Using single-strand conformation polymorphism analysis of a mouse backcross panel, the gene encoding cdc42 has been localized to distal chromosome 4.

Congenital dyserythropoietic anemias.

The congenital dyserythropoietic anemias (CDAs) are a group of relatively rare inherited anemias that share in common ineffective erythropoiesis and morphologic abnormalities of mature red blood cells and their precursors. Three major types of CDA and a number of variants have been described. The diagnosis and categorization of these disorders are facilitated by microscopic examination of the blood and bone marrow and by serologic testing. Management of patients currently consists of observation and supportive care. Because patients with CDAs may be at significant risk for secondary hemochromatosis, they require monitoring for this condition. Splenectomy may be of benefit in certain cases in which the anemia is particularly severe. Over the past few years advances have been made in understanding the pathogenesis of these disorders, and it now appears that CDA II results from enzymatic defects in the cellular glycosylation pathway.

Simultaneous addition of EGF prolongs the increase in cytosolic free calcium seen in response to bradykinin in NRK-49F cells.

The calcium-sensitive fluorescent indicator fura-2 and a microscope equipped for rapidly changing excitation wavelengths were used to look at the effects of growth factors on cytosolic free calcium ([Ca2+]i) in NRK-49F cells. In these cells bradykinin induced a rapid increase in [Ca2+]i, which generally decayed to near basal [Ca2+]i within 3 minutes. The initial rise in [Ca2+]i in response to bradykinin was relatively independent of extracellular calcium; however, the decay to basal [Ca2+]i was more rapid in the absence of extracellular calcium. Measurements made on individual cells showed a heterogeneity in the response to bradykinin. Epidermal growth factor (EGF) had no effect on [Ca2+]i in NRK-49F cells when added alone in the presence of extracellular calcium. Simultaneous addition of bradykinin and EGF produced a more prolonged increase in [Ca2+]i than bradykinin alone. The prolongation was dependent on the presence of extracellular calcium and did not occur in its absence. Transient increases in [Ca2+]i occurring after the initial peak were occasionally seen in these cells. Our results indicate that there is rapid interaction between the signaling mechanisms for bradykinin and EGF. When this occurs, one effect is the transport of calcium into the cell from the extracellular environment, causing a more prolonged rise in [Ca2+]i. This effect occurs within 1 minute after combined addition of bradykinin and EGF.

Usefulness of bone marrow examination in the evaluation of unexplained fevers in patients infected with human immunodeficiency virus.

The diagnostic usefulness of 69 bone marrow examinations (BMEs) performed for evaluating unexplained fever in 65 persons who were infected with human immunodeficiency virus was retrospectively compared with the usefulness of other diagnostic modalities used to investigate the cause of fever. An etiology for the fever was identified by BME in 22 of the 69 cases (diagnostic yield, 32%). In 19 of these 22 cases, the same diagnosis had been made by another diagnostic modality, but the diagnosis was made by BME as rapidly or sooner in 10 of the 19 cases and was made exclusively by BME in the three other cases. We suggest that BME is indicated when a diagnosis is urgently sought or when an evaluation with other diagnostic modalities has been unsuccessful.

Targeting of a heterologous protein to a regulated secretion pathway in cultured endothelial cells.

The stimulation of regulated exocytosis in vascular endothelial cells (EC) by a variety of naturally occurring agonists contributes to the interrelated processes of inflammation, thrombosis, and fibrinolysis. The Weibel-Palade body (WPB) is a well-described secretory granule in EC that contains both von Willebrand factor (vWF) and P-selectin, but the mechanisms responsible for the targeting of these proteins into this organelle remain poorly understood. Through adenoviral transduction, we have expressed human growth hormone (GH) as a model of regulated secretory protein sorting in EC. Immunofluorescence microscopy of EC infected with GH-containing recombinant adenovirus (GHrAd) demonstrated a granular distribution of GH that colocalized with vWF. In contrast, EC infected with an rAd expressing the IgG(1) heavy chain (IG), a constitutively secreted protein, did not demonstrate colocalization of IG and vWF. In response to phorbol ester, GH as well as endogenously synthesized vWF were rapidly released from GHrAd-infected EC. By immunofluorescence microscopy, granular colocalization of GH with endogenous tissue-type plasminogen activator (tPA) was also demonstrated, and most of the tPA colocalized with vWF. These data indicate that EC are capable of selectively targeting heterologous proteins, such as GH, to the regulated secretory pathway, which suggests that EC and neuroendocrine cells share common protein targeting recognition signals or receptors.

Advillin (p92): a new member of the gelsolin/villin family of actin regulatory proteins.

A new member of the gelsolin/villin family of actin regulatory proteins was initially identified by screening an adult murine brain cDNA library with a probe for bovine adseverin. The predicted amino acid sequence of the 92 kDa murine protein p92 (advillin) is 75% homologous to villin and 65% homologous to gelsolin and adseverin. It shares a six domain structure with other gelsolin family members and has a carboxy-terminal headpiece, similar to, yet distinct from, villin. Northern blot analysis shows a high level of mRNA expression in murine uterus and human intestine. In situ mRNA analysis of adult murine tissues demonstrates that the message is most highly expressed in the endometrium of the uterus, the intestinal lining, and at the surface of the tongue. In murine embryonic development, strong expression of the message is observed by day 14.5 in dorsal root ganglia and trigeminal ganglia. Expression is also noted at day 16.5 in cerebral cortex. We propose that p92 (advillin) has unique functions in the morphogenesis of neuronal cells which form ganglia, and that it may compensate to explain the near normal phenotype observed in villin-deficient mice.

An information system to improve the safety and efficiency of chemotherapy ordering.

We developed a computer application to support the ordering of chemotherapy. Key goals were to guard against errors in chemotherapy ordering and dosing, to, coordinate the outpatient and inpatient chemotherapy services, and to support the overall process flow of a chemotherapy cycle. In a six-month period, 512 daily-dose and 386 weekly-dose warnings were generated; 167 (19%) resulted in a cancellation or re-evaluation of the dose. The system has been well accepted, and has helped to coordinate the efforts of the many members of the oncology care team.

Chloramphenicol-induced mitochondrial dysfunction is associated with decreased transferrin receptor expression and ferritin synthesis in K562 cells and is unrelated to IRE-IRP interactions.

Chloramphenicol is an antibiotic that consistently suppresses the bone marrow and induces sideroblastic anemia. It is also a rare cause of aplastic anemia. These toxicities are thought to be related to mitochondrial dysfunction, since chloramphenicol inhibits mitochondrial protein synthesis. We hypothesized that chloramphenicol-induced mitochondrial impairment alters the synthesis of ferritin and the transferrin receptor. After treating K562 erythroleukemia cells with a therapeutic dose of chloramphenicol (10 microg/ml) for 4 days, there was a marked decrease in cell surface transferrin receptor expression and de novo ferritin synthesis associated with significant decreases in cytochrome c oxidase activity, ATP levels, respiratory activity, and cell growth. Decreases in the transferrin receptor and ferritin were associated with reduced and unchanged message levels, respectively. The mechanism by which mitochondrial dysfunction alters these important proteins in iron homeostasis is not clear. A global decrease in synthetic processes seems unlikely, since the expression of the cellular adhesion proteins VLA4 and CD58 was not significantly decreased by chloramphenicol, nor were the message levels of beta-actin or ferritin. The alterations were not accompanied by changes in binding of the iron response protein (IRP) to the iron-responsive element (IRE), although cytosolic aconitase activity was reduced by 27% in chloramphenicol-treated cells. A disturbance in iron homeostasis due to alterations in the transferrin receptor and ferritin may explain the hypochromic-microcytic anemia and the accumulation of nonferritin iron in the mitochondria in some individuals after chloramphenicol therapy. Also, these studies provide evidence of a link between mitochondrial impairment and iron metabolism in K562 cells.

Pulmonary embolism and deep venous thrombosis during pregnancy or oral contraceptive use: prevalence of factor V Leiden.

Activated protein C resistance caused by factor V Leiden mutation is the most common inherited cause of an underlying predisposition to pulmonary embolism (PE) and deep venous thrombosis (DVT). We studied the frequency of the factor V Leiden mutation in 50 women who had PE and/or DVT during or after pregnancy or during oral contraceptive use. Ten (20%; 95% CI 10% to 34%) of the 50 women were heterozygous for the mutation. First-trimester PE or DVT developed in 6 (60%; 95% CI, 26% to 88%) of the 10 women with the mutation compared with 3 (8%; 95% CI 2% to 20%) of 40 women without the mutation (p = 0.0009). These data indicate that the factor V Leiden mutation is an important risk factor for PE or DVT during pregnancy (especially the first trimester), after pregnancy, or during oral contraceptive use.